With the press of scientific research in the area of cancer diagnosis and the relatively recent developments involving antibodies in general and monoclonal antibodies specifically, significant advancements in the field of immunoassays have been and are continuing to be made. For instance, the development of monoclonal antibodies, i.e. a purified population of specifically reactive biological molecules, permits specific targeting for particular antigens by virtue of their manner of production and selection. This allows the development and identification of antigenic maps which describe and characterize both normal and abnormal cells. The invention described later is capable of employing both monoclonal and polyclonal antibodies.
For instance, it has been discovered that the presence of terminal deoxynucleotidyl transferase (TdT), an enzyme that can polymerize deoxynucleotides onto a primer in the absence of a template, serves as a useful indicator for the differential diagnosis of some hematologic malignancies and disorders. For example, TdT has been found absent in cells from non-hematologic tumors, Hodgkin's lymphoma, B cell lymphoproliferative disorders, peripheral T cell neoplasms, reactive lymphadenopathy, and acute nonlymphocytic leukemia. In contrast, non-T non-B cell acute lymphocytic leukemia, T cell acute lymphocytic leukemia, T cell lymphoblastic lymphoma and chronic granulocytic leukemia in blast crisis have been noted to contain marked TdT activity. For a discussion see: Castella et al, "Terminal Deoxynucleotidyl Transferase Activity in Non-Hematologic and Hematologic Neoplasms", Annals of Clinical and Laboratory Science, Vol. 12, No. 5:403-407 (1982). Such a differential diagnosis is particularly useful for monitoring relapses of acute lymphocytic leukemia as well as for predicting patient response to Vincristine and Prednisone therapy treatments.
Other autoimmune connective tissue diseases produce a spectrum of autoantibodies having specificity for a variety of nuclear antigens. Generally, the antinuclear antibodies (ANA hereafter) may belong to any of the classes of immunoglobulins (IgM, IgG, IgA, IgD, and IgE) and are not species-specific. ANAs often demonstrate specificity for one or several different nuclear antigens including deoxynucleoprotein, single and double stranded DNA, nucleoli including nucleolar ribonucleoprotein, histones, and saline-extractable antigens. The presence of antinuclear antibodies has long been recognized as a symptom of systemic lupus erythematosus (SLE). Other conditions that produce ANA include scleroderma, Sjogrens Syndrome, dermatomyositis, rheumatoid arthritis, polymyositis, and mixed connective tissue disease.
Consequently, it is quite clear that the development of tests for detecting the presence of specified intranuclear antigens such as TdT as well as antinuclear antibodies is of great significance in the clinical environment. One such technique for the detection of TdT is described by Kung et al in an article entitled "Terminal Deoxynucleotidyltransferase", Biological Chemistry, Vol. 251, 8:2399-2404 (4/25/76). That article describes a competitive type radioimmunoassay using .sup.125 I-labeled pure enzyme and antisera for the detection and quantitation of calf terminal-transferase. Such a method, however, disadvantageously requires radioisotopes necessitating careful handling as well as the obvious associated spectrum of risks and disadvantages entailed therewith. Further, such a method provides data in the form of nanograms of enzyme/milligram of cell protein but gives no information regarding the presence or absence of enzymes or intranuclear antigens within cells and cell types.
It is an object of the present invention to provide both methods and reagents for the determination and localization of intranuclear antigens in general and TdT specifically, within cells of a mixed cell population without relying on radioisotopes.
An indirect immunofluorescence method has also been described in the Castella et al article cited above. Although this method does provide some differential information with regard to the presence of TdT within cells, it disadvantageously is limited to microscopic preparations involving glass slides. Further, the procedure described requires an incubation of the cells with highly concentrated TdT specific antibody thereby greatly increasing expense without a concomitant increase in sensitivity, which parameters are important criteria within the typically over-burdened clinical environment.
It is an object of the present invention to substantially reduce the heretofore previously required high antibody concentrations thereby greatly reducing cost and to obtain substantially the same or greater sensitivity.
It is yet another object of the present invention to provide methods and reagents which may be used in flow cytometry apparatus such as the type described in U.S. Pat. Nos. 4,284,412 to Hansen et al, 3,705,771 to Friedman et al, 4,202,625 to Weiner et al, 4,284,355 to Hansen et al and 4,325,706 to Gershman et al, all of which are incorporated herein by reference, whereby large numbers of cells in a sample containing a mixed population of cells may be analyzed very rapidly and with high accuracy by utilizing hydrodynamic focusing, focused light sources and light scatter and/or fluorescence detectors with appropriate electronic analysis.
Another article by Cibull et al, "Evaluation of Methods of Detecting Terminal Deoxynucleotidyl Transferase in Human Hematologic Malignancies", A. J. C. P. Vol. 77, No. 4:420-423 (April 1982) provides the results of a correlation study comparing the specificity and sensitivity of an immunofluorescence technique, such as that of Kung et al described above, with a biochemical assay technique for determining enzymatic activity. The reference does indicate that although the radiometric assay and the immunofluorescence method do not reflect equivalent data, each method provides useful diagnostic criteria despite the fact that the significance of the difference in data is still not completely understood.
Typically, the conventional fluorescent methods for detecting either TdT such as those supplied by BRL (Bethesda Research Laboratories) or for detecting ANA such as the Nidas Test Kit available from Fisher Diagnostics, have uniformly relied on significant volumes and high concentrations of antibody reagents in order to overcome sensitivity limitations. These limitations have heretofore been the result of relatively slow and diminished cellular uptake.
It is yet another object of the present invention to provide methods for detecting intranuclear antigens, intracellular antigens, and ANAs which overcome limitations heretofor characteristic of conventional methods. Such a goal is particularly laudatory in view of the, as yet, relatively expensive and limited supply of antibodies having the required degree of specificity and affinity or binding capacity. The reduction in cost of such test kits is therefore a related object of the present invention.